The present invention relates to products and processes for amplification of nucleic acids and for expression of nucleic acids in plants.
Biomolecules such as complex proteins and nucleic acids have shown their value as products in such diverse fields as medical diagnostics/therapeutics and agriculture. Given the potential value of the products, it is not surprising that the efficient large-scale production of these biomolecules remains an active area of inquiry in biotechnology. Existing methods vary in their capacities to produce a desired biomolecule and these methods vary in terms of the costs required to achieve those levels of production. Production methods exhibiting lowered per unit production costs would provide improved approaches to the generation of biomolecules.
One strategy for improving biomolecule production is directed to maximizing the yield of the desired biomolecule. One type of approach to maximizing yield has focused on minimizing the loss of biomolecules in the production process. Recovery of active biomolecules such as polypeptides is frequently lowered due to chemical events (e.g., oxidation) and biochemical events (e.g., nuclease- or protease-mediated degradation). Thus, some production methods address these losses by adding, e.g., an anti-oxidant (xcex2-mercaptoethanol or dithiothreitol) or an inhibitor of either nucleases (e.g., vanadium complexes) or proteases (e.g., phenylmethylsulfonylfluoride). Alternative approaches modify the expressed biomolecule to confer increased stability (e.g., alkylation). However, all of these interventions are costly.
Other strategies to lower unit costs have concentrated on improving the gross yield of a desired biomolecule. Towards that end, refinements to production methods have been developed that increase the raw production of, e.g., a polypeptide by improving the genetic expression of the desired product. For example, a variety of recombinant techniques have been used to operatively link a coding region for a desired polypeptide to a strong (i.e., active) expression control signal such as a promoter An alternative approach exploits the copy number effect by amplifying copies of a coding region to provide more xe2x80x9csubstratexe2x80x9d for expression. For example, Schimke et al., J. Biol. Chem. 263:5989-5992 (1988), disclosed a technique that involved a recombinant construct that linked the Dihydrofolate Reductase (DHFR) gene to a coding region of interest. Because DHFR quantity is positively correlated to resistance to the anti-cancer drug methotrexate, challenging cells containing the construct with methotrexate selected for cells that had amplified the construct, thereby producing more DHFR and more of the desired expression product.
Another strategy for amplification has been the development of transformation systems that result in multiple copies of an introduced DNA sequence integrating into a host genome. The ribosomal DNA (i.e., rDNA) locus is an attractive target for integration since it provides a promisingly high number of target sites for integration, extending upwards from approximately 100 copies in eukaryotes. Lopes et al., Gene 79:199-206 (1989). Some multiple copy transformation systems targeting rDNA have been established using unicellular eukaryotes (Tondravi et al., Proc. Natl. Acad. Sci. (USA) 83:4369-4373 (1986); Lopes et al., (1989); Tsuge et al., Gene 90:207-214 (1990)) and cultured mouse fibroblasts (Hemann et al., DNA and Cell Biol. 13:437-445 (1994), incorporated herein by reference. Plasmid vectors containing sequences homologous to an rDNA region dramatically increased the transformation efficiency of the yeast S. cerevisiae and the phytopathogenic fungus Alternaria altenata. Yeast cells transformed with an rDNA-based expression vector containing the homologous gene for pliosphoglycerate Vase (PGK) and a heterologous gene for thaumatin were shown to carry 100-200 copies of the introduced sequences per cell. Lopes et al., (1989). Under optimized conditions, the level of PGK in transformed cells was about 50% of total soluble protein. The yield of thaumatin in transformants exceeded by a factor of 100 the level of thaumatin observed in transformants carrying only a single thaumatin gene.
Hemann et al., (1994) extended this approach by developing a high-copy expression system for mouse L fibroblasts, thereby overcoming a variety of biochemical deficiencies in enzyme-deficient cell lines. The system relied on the inclusion of an amplification promoting sequence (muNTS1), derived from the nontranscribed spacer region of murine rDNA, in the transformation vector. Wegner et al., Nucl. Acids Res. 17:9909-9932 (1989), incorporated herein by reference. The muNTS1 was originally isolated from mouse rDNA by screening with a vector containing a truncated promoter driving the expression of a thyymidine lanase gene. The high copy number amplification was achieved by the 370-bp amplification promoting element (muNTS1). Holst et al., Cell 52:355-365 (1988). Under these conditions, muNTS1 promoted amplification of the integrated vector. Copy number determination showed that muNTS1 mediated a 40- to 800-fold amplification of the vector DNA in transfected L cells. Wegner et al. (1989). The high copy number resulted in increased expression levels of the reporter gene. Further, muNTS1 was reported to promote vector amplification without selective pressure for amplification. Meyer et al., Gene 129:261-268 (1993).
These approaches to biomolecule production have been implemented using host cells of fungal or animal origin. Frequently, a commercially desirable biomolecule is a eukaryotic (e.g., human) polypeptide requiring post-translational modifications such as glycosylation, phosphorylation, etc., that a yeast host cell cannot provide. The vast majority of the unmodified analogs of these desirable polypeptides lack activity and are of little commercial value. In contrast, animal cells are eukaryotic cells typically capable of properly modifying an expressed polypeptide such as a human polypeptide. Animal cells are heterotrophic cells, however, requiring the costly inputs of energy and nutrients. Additionally, animal cell and tissue cultures require the costly maintenance of sterile conditions to prevent destructive contamination. With respect to transgenic animals, sterile conditions may not need to be maintained but, in addition to ethical concerns, the use of transgenic animals as polypeptide factories requires the costly raising of the animals and the costly isolation of the desired biomolecule.
Therefore, a need continues to exist in the art for biomolecule production methods that optimize the yield of a biomolecule, preferably in active form.
The products and methods of the present invention satisfy the aforementioned need in the art by providing a radically different approach to the production of biomolecules which relies on plant cells that have a biology far removed from animal, fungal or bacterial cells. Plant cells are photoautotrophic cells which typically do not require an energy input. Moreover, plant cells typically require a minimal nutrient input in terms of number, quantity and cost. Thus, plant cells provide cost advantages over the more familiar animal, fungal and bacterial host cell environments. The products and methods of the invention are designed to amplify, and optionally improve the expression of, nucleic acids in the cells of a plant. The invention reals the advantage of lowered costs resulting from reduced host cell requirements for energy and nutrients.
The present invention provides novel plant polynucleotides specifying Amplification Promoting Sequences (APSs) which are active in plants to amplify an adjacent target nucleic acid. For target nucleic acids encoding an expressible product, the plant polynucleotides according to the invention may be capable of improving expression beyond expectations focused solely on copy number. Preferred APSs of the invention comprise A-T rich (preferably 80% or more) rDNA intergenic polynucleotide sequences or subsequences derived from (or based on) e.g., tobacco (Borisjuk et al., Plant Mol. Biol. 35:655-660 (1997); SEQ ID NO:1 and SEQ ID NO:2), L. esculentum (Perry et al., Mol. Gen. Genet. 221:102-112 (1990); SEQ ID NO:4), and S. tuberosum (Borisjuk et al., Plant Mol. Biol. 21:381-384 (1993); SEQ ID NO:3). Presently most preferred plant APSs comprise the N. tabacum-derived polynucleotide set out in SEQ ID NO:1 and plant polynucleotides which hybridize under stringent conditions to the complement of the sequence set forth in SEQ ID NO:1.
Also provided by the present invention are nucleotide segments comprising a plant APS and an adjacent target nucleic acid to be amplified, preferably located within the segment within 3000 nucleotides from the APS. Novel nucleotide segments of the invention may also comprise a plant APS and a homologous recombination locator polynucleotide functional to provide for site-specific insertion of the APS within another polynucleotide, e.g., a plant host chromosome or vector. Preferred novel nucleotide segments comprise vectors, including Agrobacterium-derived plant transformation vectors.
In another aspect of the invention, plants and plant host cells are provided which are stably transformed or transfected with plant-derived and non-plant derived APSs functional to amplify adjacent nucleic acids within the plants or plant cells. Such adjacent nucleic acids may be coding sequences (e.g., RNA and/or protein coding) or may be expression regulatory sequences. The present invention correspondingly provides novel methods for amplifying target nucleic acids within plants, which methods optionally include isolation of products coded thereby.
Yet another aspect of the invention is a method for enhancing mRNA expression of a target nucleic acid comprising the step of contacting a plant host cell under conditions suitable for transforming or transfecting the cell with an isolated polynucleotide specifying a plant-active Amplification Promoting Sequence (APS), the polynucleotide selected from the group consisting of a) a polynucleotide comprising the sequence set forth in SEQ ID NO:1 or a fragment thereof; and b) a polynucleotide which hybridizes to a polynucleotide having the complement of the sequence set forth in step a), the hybridization occurring in 3xc3x97SSC, 20 mM NaPO4 (pH 6.8) at 65xc2x0 C. with washing in 0.2xc3x97SSC at 65xc2x0 C. The methods are particularly suited for the use of target nucleic acids that encode a polypetide; the methods may further comprise the step of recovering the expression product of said target nucleic acid. In addition, the isolated polynucleotide used in the methods may further comprise a promoter.
Numerous other aspects and advantages of the present invention will be apparent upon consideration of the following detailed description, including the drawing.